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Vitamin D
Now moving on to vitamin D--vitamin D is the other major regulator. It is really a hormone. The only reason it is called a vitamin is because its source is in part dietary. Otherwise, it behaves like a hormone, it is tightly regulated, it has enzymes to control its behavior, and its origination is generally from either sunlight or your diet. It is then converted into vitamin D2 at the liver. It is converted in the kidney to the 1-alpha-dihydroxy vitamin D3. But it is also converted to 25 and 24-25 vitamin D.

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Source: Martin KJ et al, Am J Kidney Dis. 1998 Oct;32(2 Suppl 2):S61-6.

Analogs of Vitamin D
There are many, many new analogs of calcitriol that are being developed for treatment of immune dysfunction, psoriasis, as well as cancers and renal failure. We will talk more about these tomorrow. Essentially what they are doing is altering this part here, which allows a little bit difference in its binding effects at different target tissues.

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Pharmacokinetics of Vitamin D
Over 40 metabolites of calcitriol or 1,25 dihydroxy vitamin D3 exist. Clearly the most important is this calcitriol. The second most abundant form is 24-25, and we still don't really know what it does. It is very metabolically active with the regulatory key in the kidney: the 1-alpha- hydroxylase enzyme in the proximal tubule. That is why there is so much derangement of calcitriol in renal failure.

The intestinal concentration peaks just one to two hours after an IV injection. As we will show tomorrow, that is probably why there really isn't a difference between oral and IV calcitriol in terms of its gut effects. It is catabolized principally through a 24- hydroxylase with biliary elimination, and the half-life normally is about 14 hours. In renal failure, it can extend up to about 20.

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1-alpha-hydroxylase
This is the enzyme in the kidney that converts the 25 to the 1,25 vitamin D. As you can see, just about anything that you would ever associate with bone growth--homeostasis regulates this enzyme. In fact, the mechanism by which almost everything controls calcium in vitamin D metabolism is through this enzyme in the kidney, which obviously is messed up in renal failure.

So the factors that control the 1-alpha-hydroxylase, the main factors, are parathyroid hormone, which stimulates it; and this is the way that PTH shuts itself off. PTH does not shut itself off directly at the parathyroid gland, it does it indirectly by regulating this enzyme, which then goes back to the gland and shuts it off. Low calcium levels stimulate this; low phosphorus levels stimulate this; it controls itself; calcitonin has a weak effect here; estrogen, prolactin, growth hormone and placental lactogen also regulate it here.

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Vitamin D receptor localization
The interesting thing about calcitriol is that while clearly the major effect is on calcium and phosphorus homeostasis, the vitamin D receptor is localized in just about every tissue in the body. So that brings up a lot of issues. Why is it there? Does it really have an importance there?

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Intracellular actions of vitamin D
The mechanism by which calcitriol affects vitamin D is that the calcitriol comes into the cell membrane. It freely diffuses it. In the cell it binds to the vitamin D receptor, the VDR. The VDR then complexes with the retinoic acid receptor to form a heterodimer that binds to something called the vitamin D response element of genes. To date there are over 49 genes that have a vitamin D response element. Important ones, obviously, are all of those that control bone, but also those that control early gene expression-- c-myc, c-fos, and other early regulators. That is why it has been thought that it probably can be altered to enhance these effects and turn off some cellular proliferation and use it in cancer.

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Mechanisms of action of calcitriol
The mechanisms of calcitriol are two fold. One is genomic, mediated through the VDR as I just went through. The other is non-genomic effects, which is membrane interaction. Calcitriol activates voltage- dependent calcium channels. It increases intracellular calcium. There are two binding proteins, calbindins, 9 kD in the intestine and 28 kD in the kidney that facilitate the cellular uptake of calcitriol.

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Target tissue effects of calcitriol
Now the target tissue effects of calcitriol is predominantly that it enhances calcium absorption from the gut, inhibits PTH secretion and enhances bone remodeling. We are still not 100 percent sure what it does in bone remodeling, but it is probably very important in terms of early recruitment and differentiation of osteoblasts and osteoclasts. As I mentioned, it also controls differentiation and proliferation in other cells and implicated in at least partial control of the cell cycle.

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Calcitonin
The third hormone that controls calcium and phosphorus is calcitonin, and it gets one slide in this presentation. It is a 32-amino acid protein produced in the thyroid C cells and it is really of no physiologic relevance in humans. However, the salmon calcitonin in humans has a much greater affinity to the calcitonin receptors. That is why all of the pharmacologic preparations are salmon. There are receptors for calcitonin located in the kidney, in the osteoclasts, and in the brain. And in fact, calcitonin is the only osteoporotic agent that has some pain relief because it does bind to opioid receptors and can give some patients some pain relief. But the net effect is to lower calcium and phosphorus, but again unknown physiologic relevance.

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Intestinal absorption of calcium
Let's move on to the target tissues. The first is intestine. This is the intestinal absorption versus the dietary intake for calcium, magnesium, and phosphorus. Phosphorus is directly related to what you eat. What you eat gets absorbed. Phosphorus regulates its own absorption at the gut. Calcium, on the other hand--the shape of the curve right here tells you that it is both partially actively mediated and partially passive. And as I mentioned earlier, you have this obligatory secretion so that if your dietary intake is really low, you are going to lose calcium.

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Calcium absorption in the gut
At the level of the gut--this is the enterocyte, calcitriol stimulates the uptake of calcium; calcium then goes into the cytosolic pool and is extruded out through a calcium ATP-ase or through a sodium-calcium exchanger. It also is transported paracellularly down its concentration gradient.

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Calcium and intestinal calcium transport
The mechanism by which calcitriol enhances this is the facilitation of a calcium by inserting calcium channels into the membrane. It also increases calbindin, which then facilitates and ferries that calcium across the cell. It increases the calcium magnesium ATP-ase, and it facilitates paracellular transport. So it works here, here, here, and here; which is why when you are vitamin D deficient, you don't absorb much calcium.

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Factors affecting intestinal calcium absorption
Other factors that increase or decrease intestinal calcium absorption include parathyroid hormones, indirect via its mechanism ... stimulation of 1-alpha-hydroxylase; a low calcium diet; growth; lactation; pregnancy; estrogen--all of these work via the 1-alpha- hydroxylase enzyme. So this list in renal failure is very different.

Things that are important to know in terms of decrease is that normally with aging, you go into neutral calcium balance in terms of what you absorb from the gut, neutral or negative. We don't really know exactly why that is, but the absorption definitely goes down. Glucocorticoids--all your renal transplants and your membranous patients are all decreasing the calcium absorption at the gut. The other two are oxalate and phytate, and these bind the calcium in the gut and prevent absorption, similarly iron.

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Phosphorus transport in the gut
Now phosphorus transport at the enterocyte occurs via sodium phosphate cotransporters in the lumen. In addition, phosphorus directly regulates the insertion of these cotransporters. Calcitriol does similarly. The phosphorus is then extruded out the basolateral membrane through phosphate transporters.

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Factors affecting intestinal phosphate absorption
The factors that affect intestinal phosphorus absorption include calcitriol; alkaline pH, which enhances the sodium phosphate cotransport affinity, but it is self-limiting; phosphorus depletion itself enhances the uptake, probably again through the 1-alpha- hydroxylase enzyme in the kidney. A high phosphorus diet though can self-increase phosphorus absorption, primarily through paracellular pathways, but also by basically inserting more sodium phosphate cotransporters.

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Renal calcium transport
Renal handling, the second target organ. Compared to the intestine, the renal tubule reabsorbs 40-fold more calcium per day. We know how to show those GI docs--our kidneys do a lot more work. The bulk of the reabsorption is in the proximal tubule, but the hormonal regulation is distally, a fairly common theme for renal handling. The ultrafiltered plasma/calcium ratio is basically equivalent to that which is not bound to albumin.

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Source: Juppner H and Schipani E. Curr Opin Nephrol Hypertens. 1996 Jul;5(4):300-6. Review.

This nephron just shows you the proportion and the areas that absorb the calcium. The majority is absorbed in the proximal tubule, the next most common is in the thick ascending limb, and then some component is distal. The mechanism of transport in the proximal convoluted tubule is primarily paracellular. In the thick ascending limb, it is about a 50/50 mix. And in the distal tubule, it is all active, which is why it is regulated there.

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Calcium transport in different parts of the nephron
The renal calcium tubule proximal tubule transport parallels salt and water, so it is almost certainly solvent drag and electrochemical gradient. That is why your little old lady who shows up in the emergency room very volume depleted has an elevated calcium. If the calcium doesn't go up in a patient, they are probably not profoundly volume depleted.

We don't really know how that works. However, parathyroid hormone, calcitonin, volume expansion, insulin and phosphorus depletion inhibit this reabsorption.

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Calcium transport in the thick ascending limb
In the Loop of Henle, there is no role in the thin descending or ascending limbs. But in the thick ascending limb, the majority is paracellular through the gradient established by the sodium potassium-2 chloride transporter, and the rest is transcellular. And somehow PTH affects that, and we are not really sure how. The calcium sensing receptor is also most abundantly expressed here.

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Source: Hebert SC and Brown EM. Curr Opin Nephrol Hypertens. 1996 Jan;5(1):45-53. Review.

So here is your thick ascending limb, apical and the basolateral site. Your sodium potassium-2 chloride exchanger facilitates uptake and then potassium is recycled out, creating a positive lumen. That facilitates the paracellular transport of calcium. This is why a loop diuretic, which blocks this transporter, is used to treat hypercalcemia. It blocks this paracellular transport. In addition, the calcium sensing receptor is on the basolateral membrane. We are not 100 percent sure what it does here except that it is important because it binds to this receptor, changes the cell signaling mechanism and inhibits normal sodium chloride uptake. In doing so in the thick ascending limb, it kind of ruins that countercurrent multiplication that washes out the gradient, and this is probably why you get, in part, the polyuria with hypercalcemia.

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Source: Friedman PA and Gesek FA. Am J Physiol. 1993 Feb;264(2 Pt 2):F181-98. Review.

Calcium transport in the distal tubule
Now in the distal tubule, really the cortical collecting duct in the distal tubule, there is a very active control of calcium. This is primarily through a calcium/sodium exchanger. We use this to treat patients with kidney stones who are hypercalciuric. So what we give them is thiazide diuretics, which block the sodium/chloride transporter, and that facilitates this exchanger because more sodium wants to go into the cell, so you reabsorb your calcium.

But you can also do this with amiloride, much less studied but it is helpful in kidney stones by blocking this channel and having the same net effect.

So the combination, such as Moduretic-TM, where you get a little thiazide and amiloride, is useful in stones, particularly in somebody who has a problem with K wasting from the thiazide.

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PTH effects on renal calcium transport
Now the PTH clearly affects renal calcium transport. It enhances GFR, it has a controversial effect on the proximal tubule, but the main effect is in the thick ascending limb in the distal tubule. The exact mechanism is not clear, although it probably enhances sodium-calcium exchanger and the calcium ATP-ase.

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Other regulators of renal calcium transport
Other regulators include a high-salt diet. We don't really understand this, but it is a very real phenomenon, particularly when you take care of kidney stone patients. If they eat a lot of salt, then you get very little benefit of the thiazide diuretic. In fact, for every 25 to 50 mg or mEq of sodium a patient can drop in their urine, they can lower the urine calcium significantly. This is very important, and one of the key reasons for failure of medical therapy of stones is a high-salt diet.

Some adaptation does occur with calcium intake, and phosphate depletion stimulates calcium excretion, as well. Increased dietary protein can increase excretion, which again in stone formers is a reason you want them to decrease their protein intake.

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Miscellaneous regulators--all of these--metabolic acidosis is important; calcitonin works; glucocorticoids enhance excretion directly by the tubules--not completely understood; insulin enhances calcium excretion. The vitamin D receptors, located along the entire nephron, but we don't know exactly what it does, but it is very important.

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Source: Kurokawa K. Kidney Int. 1987 Nov;32(5):760-71. No abstract available.

Urine calcium excretion as a function of serum calcium
So as your serum calcium increases--this is your urine calcium, and this is in rats, but if you have D deficient and PTH deficient, here is your curve. If you give this rat vitamin D or you give them just PTH alone, you shift it to the right. If you give them both vitamin D and PTH, you shift it even further. So clearly there is some interaction of the PTH and the vitamin D receptor at the level of the kidney.

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Summary of calcium transport in the kidney
This just kind of summarizes all the things that affect calcium excretion: hypercalcemia, hypocalcemia, changes in GFR, volume status, loop diuretics and the calcium and magnesium concentration itself, PTH, thiazide, amiloride, and then phosphate changes.

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Source: Hebert SC. Kidney Int. 1996 Dec;50(6):2129-39. Review. No abstract available.

Effect of calcium-sensing receptor on calcium transport
As I mentioned, the increased calcium, or really magnesium as well, binds to that basolateral calcium sensing receptor. This decreases sodium chloride absorption in the thick ascending limb, which decreases the urinary concentrating ability and changes the lumen positive voltage, decreases that. The net effect is that you get rid of the calcium in a very dilute urine, which is probably a safety mechanism to inhibit stones. In addition, the calcium sensing receptor may also directly affect the aquaporin channels in the distal tubule, again giving you polyuria with hypercalcemia.

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Renal phosphorus transport
Moving on to renal phosphorus transport, phosphorus is freely filtered at the glomerulus. The renal tubules reabsorb the vast majority in the proximal tubule, 30 percent in the distal tubule. The ability of the tubules to reabsorb phosphorus becomes saturable at a serum phosphorus of about 6 g/dL, the TM for phosphorus. You see a lot of physiology studies that look to try to see if this is altered. Clearly there is a direct correlation between GFR and TM phosphorus. That little nomogram is your handout, but we won't go into that.

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Phosphorus absorption in the kidney: Proximal tubule
The mechanism of phosphorus absorption in the tubules is almost identical to that in enterocytes. Sodium phosphate cotransporters-- they are stored in an endocytotic compartment here. In the presence of changes in phosphorus, they are inserted in here to increase excretion or reabsorption.

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Again, phosphorus enters the brush border through the sodium phosphate cotransporter and exits the cell down the electrochemical gradient established by the sodium potassium ATP-ase. The acute adaptation to a low phosphorus diet is the insertion of more stored sodium phosphate transporters. Chronically it will turn on gene synthesis. In fact, you could get rid of PTH and get rid of vitamin D and if you just had a normal kidney, you could regulate serum phosphorus. It is much more important in its own control than is calcium. But PTH and vitamin D are clearly important, as well.

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Phosphorus handling in the kidney: Distal tubule
In the distal tubule, about 30 percent is reabsorbed, although that is somewhat controversial. And that is inhibited by PTH. So the mechanism by which you get the phosphorus losses in urine via PTH is all distal tubule. We don't know exactly how.

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Factors affecting renal phosphorus handling
This again is a nice slide, I think, that just summarizes all the factors that affect phosphorus excretion: the values themselves or the blood levels themselves, the dietary phosphorus intake, PTH a little bit here, glucocorticoids have a key effect in terms of inhibiting phosphorus reabsorption, low phosphorus diet, and PTH.

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Source: Drezner MK. Kidney Int. 2000 Jan;57(1):9-18. Review.

Phosphatonin
One other interesting thing that has developed a lot of information recently is something called phosphatonin. This is an elusive compound. It has not really been identified. It is probably a family of compounds that appear to have a receptor in the renal tubule cell and thereby turn on the sodium phosphate cotransport. So phosphatonin normally is helpful in determining the amount of phosphate excretion.

This substance can be secreted in certain malignancies, giving you a phosphorus-wasting picture. In addition, in X-linked hypophosphatemic rickets, there is a defect in something called the PHEX gene, which is an endopeptidase that sits at the osteoblast membrane. That is responsible for digesting this phosphatonin. This is somewhat hypothetical yet, but it is important in digesting the phosphatonin. So if you have a defect in this gene, you have excessive phosphatonin, and that is why you get the phosphorus wasting in the X-linked hypophosphatemic rickets. So again, PHEX has been identified and clearly is the culprit gene in that disease.

References

1. Cogan MG. Fluids and Electrolytes: Physiology and Pathophysiology. Appleton and Lange. 1991.

2. Coe FL. Disorders of Bone and Mineral Metabolism. Lippincott Williams and Wilkins. 1982.

3. Brown EM. Four-parameter model of the sigmoidal relationship between parathyroid hormone release and extracellular calcium concentration in normal and abnormal parathyroid tissue. J Clin Endocrinol Metab. 1983 Mar;56(3):572-81.

4. Brown EM, Pollak M, Seidman CE, Seidman JG, Chou YH, Riccardi D, Hebert SC. Calcium-ion-sensing cell-surface receptors. N Engl J Med. 1995 Jul 27;333(4):234-40. Review. No abstract available.

5. Naveh-Many T, Friedlaender MM, Mayer H, Silver J. Calcium regulates parathyroid hormone messenger ribonucleic acid (mRNA), but not calcitonin mRNA in vivo in the rat. Dominant role of 1,25-dihydroxyvitamin D. Endocrinology. 1989 Jul;125(1):275-80.

6. Almaden Y, Hernandez A, Torregrosa V, Canalejo A, Sabate L, Fernandez Cruz L, Campistol JM, Torres A, Rodriguez M. High phosphate level directly stimulates parathyroid hormone secretion and synthesis by human parathyroid tissue in vitro. J Am Soc Nephrol. 1998 Oct;9(10):1845-52.

7. de Francisco AL, Cobo MA, Setien MA, Rodrigo E, Fresnedo GF, Unzueta MT, Amado JA, Ruiz JC, Arias M, Rodriguez M. Effect of serum phosphate on parathyroid hormone secretion during hemodialysis. Kidney Int. 1998 Dec;54(6):2140-5.

8. Brown AJ, Ritter CS, Finch JL, Slatopolsky EA. Decreased calcium-sensing receptor expression in hyperplastic parathyroid glands of uremic rats: role of dietary phosphate. Kidney Int. 1999 Apr;55(4):1284-92.

9. Juppner H, Schipani E. Receptors for parathyroid hormone and parathyroid hormone-related peptide: from molecular cloning to definition of diseases. Curr Opin Nephrol Hypertens. 1996 Jul;5(4):300-6. Review.

10. Martin KJ, Gonzalez EA, Gellens ME, Hamm LL, Abboud H, Lindberg J. Therapy of secondary hyperparathyroidism with 19-nor-1alpha,25-dihydroxyvitamin D2. Am J Kidney Dis. 1998 Oct;32(2 Suppl 2):S61-6.

11. Favus MJ, ed. Primer on the Metabolic Bone Diseases and Disorders of Mineral Metabolism. Lippincott Williams and Wilkins. 1996.

12. Hebert SC, Brown EM. The scent of an ion: calcium-sensing and its roles in health and disease. Curr Opin Nephrol Hypertens. 1996 Jan;5(1):45-53. Review.

13. Friedman PA, Gesek FA. Calcium transport in renal epithelial cells. Am J Physiol. 1993 Feb;264(2 Pt 2):F181-98. Review.

14. Kurokawa K. Calcium-regulating hormones and the kidney. Kidney Int. 1987 Nov;32(5):760-71. No abstract available.

15. Hebert SC. Extracellular calcium-sensing receptor: implications for calcium and magnesium handling in the kidney. Kidney Int. 1996 Dec;50(6):2129-39. Review. No abstract available.

16. Drezner MK. PHEX gene and hypophosphatemia. Kidney Int. 2000 Jan;57(1):9-18. Review.

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Topic Index:  Chronic Renal Failure -- Bone Disease Topic Index:  Clinical Nephrology -- Hypercalcemia